Cucurbitacin B Inhibits Cell Proliferation and Induces Apoptosis in Human Osteosarcoma Cells Via Modulation of the JAK2/STAT3 and MAPK Pathways

EXPERIMENTAL AND THERAPEUTIC MEDICINE 14: 805-812, 2017

Cucurbitacin B inhibits cell proliferation and induces apoptosis in human osteosarcoma cells via modulation of the JAK2/STAT3 and MAPK pathways

ZHI‑REN ZHANG1, MING‑XIA GAO2 and KAI YANG3

1Department of Orthopedics, Zhumadian Central Hospital, Zhumadian, Henan 463600; 2Department of Health Management, Dongying People's Hospital, Dongying, Shandong 257000; 3Department of Joint Surgery, Zhengzhou Central Hospital Affiliated to Zhengzhou University, Zhengzhou, Henan 450007, P.R. China

Received October 20, 2015; Accepted February 27, 2017

DOI: 10.3892/etm.2017.4547

Abstract. Osteosarcoma (OS) is the most commonly diagnosed Introduction tumor of the bones in children and young adults. Even with conventional therapies the 5‑year survival rate is ~65% in Osteosarcoma (OS) a primary sarcoma of the bones that patients with OS. Considering the side effects and aggres- primarily affects children and adolescents accounting for ~5% siveness of malignant bone tumors, research is focussing on of pediatric tumors (1). OS affects the distal long bones, the multi‑targeted strategies in treatment. Cucurbitacin B, a triter- femur and tibia (1) and is generally characterized by its local penoid compound has been demonstrated to induce apoptosis invasion of bone and soft tissues, loss of the affected extremity's in various cancer cell types. The Janus kinase 2/signal trans- functions and distant metastasis (2). Multimodal conventional ducer and activator of transcription 3 (JAK2/STAT3) signalling therapies including radiation, surgical resection and chemo- cascades and mitogen activated protein kinases (MAPK) therapy are employed to treat OS (3,4). Despite the combination signalling cascades are critical regulators of tumorigenesis. therapy approach, limited improvement has been observed in the The present study assessed the influence of cucurbitacin B 5‑year survival rate (65%) of patients with OS (5). Furthermore, on the viability and expression of MAPKs and proteins of these therapeutic approaches often lead to severe side effects the JAK2/STAT3 cascades in human OS cells (U‑2 OS). such as cardiotoxicity, hearing loss and nephrotoxicity, and Cucurbitacin B (20‑100 µM) significantly reduced cell viability may also cause drug resistance and increase the risk of local (P<0.05) and induced apoptosis, as assessed by MTT and relapse (6). Thus, improved targeted approaches are required, Annexin V/propidium iodide staining, along with inhibiting with no or less side effects. cell migration. Gelatin zymography revealed supressed activi- The Janus kinase 2 (JAK2)/signal transducer and activator of ties of matrix metalloproteinase (MMP‑)2 and 9. Furthermore, transcription 3 (STAT3) signalling pathway serves a critical role cucurbitacin B effectively upregulated the apoptotic pathway in cell survival and division. Activated JAK2/STAT3 signalling and caused the effective inhibition of MAPK signalling and cascade influences the expression of numerous proteins that JAK2/STAT3 cascades. Multifold suppression of vascular are associated with various physiological functions, including endothelial growth factor by cucurbitacin B was also observed, cell cycle regulation and apoptosis. The JAK/STAT3 pathway indicating inhibition of angiogenesis. Thus, by downregu- regulates the expression level of anti‑apoptotic proteins [B‑cell lating major pathways‑MAPK and JAK2/STAT3 and MMPs, lymphoma‑extra large (Bcl‑xL) and myeloid leukemia cell cucurbitacin B has potent anti‑proliferative and anti‑metastatic differentiation protein (Mcl‑1)] (7), cell cycle regulatory proteins effects that require further investigation with regards to cancer (cyclin D1, p21, and p27) (8) and mitochondrial apoptosis treatment. pathway related proteins [Bcl‑2 associated X (Bax), cytochrome c and caspase‑3] (9). Furthermore, STAT3 activation promotes angiogenesis by inducing vascular endothelial growth factor (VEGF) (10), and also stimulates invasion and metastasis by increasing the expression of matrix metalloproteinases Correspondence to: Dr Kai Yang, Department of Joint Surgery, (MMPs) (11). The pathway is considered as a major molecular Zhengzhou Central Hospital Affiliated to Zhengzhou University, 195 Tongbai Road, Zhengzhou, Henan 450007, P.R. China target of interest in a number of cancer types, including mela- E‑mail: [email protected] noma (12), renal carcinoma (13) and breast cancer (14). The mitogen‑activated protein kinases (MAPKs) signalling Key words: apoptosis, cucurbitacin, Janus kinase/signal transducer cascades are a large family of serine/threonine kinases that and activator of transcription pathway, mitogen activated protein control and regulate various physiological process, including cell kinases, osteosarcoma survival and apoptosis, and are also involved in tumorigenesis. The functions of MAPK signalling in cancer development are complex, as they regulate wide range of cellular responses (15). 806 ZHANG et al: CUCURBITACIN B INDUCES APOPTOSIS OF HUMAN OSTEOSARCOMA CELLS

Activated MAPK pathway stimulates cell growth or induces Measurement of cell viability. OS cells were seeded into apoptosis based on the stimuli (16,17). Numerous studies have 96‑well plates (5x105 cells/well) and incubated at 37˚C for reported that the c‑Jun N‑terminal kinases (JNK), p38 and 24 h. On reaching 70% confluence, the cells were treated extracellular signal‑regulated kinases (ERK1/2) cascades exert with different concentrations of cucurbitacin B (20, 40, 80 a vital role in regulating cytotoxic drug induced apoptosis in and 100 µM) for 24 h. Cells were then incubated at 37˚C with OS (18,19). Thus, targeting the pathway may have clinical value MTT for 4 h. The formazan crystals formed were dissolved in the treatment of OS. with dimethyl sulfoxide and the absorbance was measured at Accumulating research data indicate that plant‑derived 570 nm using a Thermo Multiskan Spectrum (Thermo Fisher compounds are much more effective in inhibiting cancer cell Scientific, Inc., Waltham, MA, USA). proliferation and inducing apoptosis (20). Phytochemicals are reported to elicit antitumor effects by inducing cellular defense Analysis of apoptosis by Annexin V assay. OS cells were incu- system, antioxidant enzymes system and also inhibition of bated at 37˚C with various concentrations of cucurbitacin B anti‑cell growth signalling and anti‑inflammatory pathways (20, 40, 80 and 100 µM) for 24 h. Following incubation, culminating in apoptosis and/or cell cycle arrest (21‑24). cells were treated with 0.25% trypsin, washed twice with Cucurbitacins were initially identified in the Cucurbitaceae ice‑cold PBS and harvested for detection of apoptosis using plant family, which includes cucumber and are also isolated an Annexin V‑FITC apoptosis detection kit (BD Biosciences, from various plant families (25). Owing to their effective phar- Franklin Lakes, NJ, USA), according to the manufacturer's macological properties, plants rich in cucurbitacins have been instructions. The cells were washed in PBS at 1x106 cells/ml widely used in traditional Chinese medicine for their analgesic, and suspended at 1x105/100 µl binding buffer (BD Biosciences) anti‑inflammatory, antimicrobial, antipyretic, antitumor activities containing FITC‑conjugated anti‑Annexin V antibody and hepatoprotective effects (25‑28). Researchers have reported (provided in the kit) and PI, and analyzed for fluorescence that cucurbitacin I may inhibit cancer cell growth by disrupting using a flow cytometer (FACSCalibur; BD Biosciences). the JAK/STAT3 signaling pathway in both in vitro and in vivo tumor models (29,30). Studies have reported that cucurbitacin B Determination of morphological changes by Hoechst inhibits the growth of various human cancer cell lines and tumor staining. Morphological changes of the U‑2 OS cells following xenografts including breast, prostate, lung, uterine cervix, liver, treatment with cucurbitacin B was determined by Hoechst skin and brain cancer (18,31,32). Considering the biological staining. Briefly, the OS cells were seeded at a density of effects cucurbitacin B, the present study aimed investigate the 5x104 cells per well in a 6‑well plate and cultured at 37˚C for effects of cucurbitacin B on human OS cells and assess whether 12 h and treated with cucurbitacin B (20, 40, 80 and 100 µM) it modulates the JAK/STAT3 and MAPK signalling pathways. for 48 h. The cells were then fixed with 4% paraformalde- hyde in PBS for 10 min, washed with PBS and stained with Materials and methods Hoechst 33258 (5 mg/l) for 15 min at room temperature in the dark. The cells were observed under a a fluorescence Reagents and chemicals. Cucurbitacin B, glutamine, microscope (Nikon Eclipse TiS coupled with NIS‑Elements RPMI 1640 medium, fetal calf serum (FCS), penicillin, strepto- imaging software; Nikon Corporation, Tokyo, Japan). The mycin and 0.25% trypsin were purchased from Sigma Aldrich; cells undergoing apoptosis were characterized by condensed Merck KGaA (Darmstadt, Germany). Antibodies against or fragmented nuclei. ERK1/2 (cat. no. 9102), phospho‑ERK1/2 (cat. no. 9101), p38 (rabbit mAb, cat. no. 8690), phospho‑p38 (rabbit mAb, cat. Wound healing assay. The influence of cucurbitacin B on no. 4511), JNK (cat. no. 9252), phospho‑JNK (mouse mAb, the cell migration of U‑2 OS cells was assessed. In brief, cat. no. 9255), MMP‑2 (rabbit mAb, cat. no. 87809), MMP‑9 5x105 cells were maintained in 10 cm Petri plates and incu- (cat. no. 852) (all from Cell Signaling Technology, Danvers, bated for 24‑72 h until they reached complete confluency and MA, USA), Bcl‑xL (cat. no. sc‑8392), Bcl‑2‑associated death were wounded using a 200 µl pipette tip. All cells in the plates promoter (Bad) (cat. no. sc‑8044), Bax (cat. no. sc‑4239), were treated with final concentrations of cucurbitacin B (25, Bcl‑2 (cat. no. sc‑509), VEGF (cat. no. sc‑4571), caspase‑9 50 and 100 µM; 3 plates for each concentration) and then (cat. no. sc‑81663), caspase‑8 (cat. no. sc‑81656), caspase‑3 were incubated at 37˚C in fresh culture medium for 24 h. The (cat. no. sc‑176260), p‑JAK2 (cat. no. sc‑34479), JAK2 (cat. migration of the cells in the wounded area was measured as no. sc‑34479), p‑STAT3 (cat. no. sc‑56747) and STAT3 (cat. previously described (33). Cell migration was determined as a no. sc‑482) (all from Santa Cruz Biotechnology, Inc., Dallas, percentage of the cell‑free area compared with the area of the TX, USA) were used for expression analysis. All other reagents initial wounded area. The fields were photographed using an used in the study were purchased from Sigma Aldrich; Merck inverted microscope (Nikon Eclipse TS2; Nikon Corporation). KGaA, unless otherwise stated. Gel zymography. Gel zymography was performed to evaluate Cell lines and culture. Human osteosarcoma cell line, U‑2 OS the activities of MMP‑2 and MMP‑9. Sample preparation was obtained from the Cell Bank of Shanghai Institute was done as previously described by Mizoguchi et al (34). of Biochemistry and Cell Biology (Chinese Academy of Samples were run on SDS‑PAGE (10%) gels that contained Sciences, Shanghai, China). The cells were cultured and main- 0.1% gelatin under non‑reducing conditions. Triton X‑100 tained in RPMI‑1640 medium supplemented with 10% FCS, (2.5%) was used to wash the gels to remove SDS, then the 2 mM glutamine, streptomycin (100 µg/ml) and penicillin gels were washed further in incubation buffer (50 mM Tris

(100 U/ml) at 37˚C in a humidified atmosphere with 5% CO2. HCl, 0.15 M NaCl, 10 mM CaCl2) (25±2˚C) for 30 min and EXPERIMENTAL AND THERAPEUTIC MEDICINE 14: 805-812, 2017 807 incubated (24 h, 37˚C). Following incubation, the gels were stained with Coomassie Brilliant Blue (1% Coomassie Brilliant Blue G‑250, 30% methanol and 10% acetic acid) for 3 h and then were destained (7% acetic acid, 40% methanol) until clear bands representing gelatinolysis were seen against dark background. By using an Atto Densitograph Software Library Lane Analyser (Atto Instruments, Inc., Rockville, MD, USA) the total activity was determined.

Western blot analysis. Following treatment with cucurbitacin B (25, 50 and 100 µM), U‑2 OS cells were washed twice with ice‑cold PBS and centrifuged at 10,000 x g for 5 min Figure 1. Cucurbitacin B reduces the cell viability of U‑2 OS cells. *P<0.05 at 4˚C. The cells were treated in ice‑cold hypotonic lysis buffer vs. control; #P<0.05 vs. 20 µM. Data are presented as mean ± standard devia- (10 mM HEPES pH 7.9, 1.5 mM MgCl2, 0.2 mM KCl, 0.5 mM tion, n=3. OS, osteosarcoma. dithiothreitol, 0.2 mM phenylmethyl‑sulphonylfluoride), vortexed for 2 min, centrifuged at 12,000 x g for 10 min at 4˚C and the supernatants were collected and assayed for protein content using the Bradford method (35). Equal amounts of protein samples (60 µg) were subjected to electrophoresis in 12% SDS‑PAGE and the bands were then transferred to nitrocellulose membranes (GE Healthcare Lifesciences, Chalfont, UK). The membranes were washed and blocked using blocking buffer (5% non‑fat dry milk/0.1% Tween‑20 in TBS) for 1 h at room temperature. They were then incubated overnight at 4˚C with respective primary antibodies followed by incubation at room temperature for 40 min with horseradish peroxidase‑conjugated‑secondary antibody (mouse mAb; Cell Signaling Technology, cat. no. 7076; dilution 1:1,000). The Figure 2. Cucurbitacin B induces apoptosis of human osteosarcoma U‑2 OS immunoreactive bands were detected by enhanced chemilu- cells. Cucurbitacin B was observed to significantly increase the apoptotic minescence procedure (GE Healthcare Lifesciences). Protein cell count and reduce cell viability in U‑2 OS cells. *P<0.05 vs. control; expression levels were normalized with the expression levels of #P<0.05 vs. 20 µM. Data are presented as mean ± standard deviation, n=3. β‑actin using anti‑β‑actin antibody (AC‑15) from Santa Cruz OS, osteosarcoma. Biotechnology, Inc. (cat. no. sc‑69879; dilution 1:1,000). The positive signals from the immunoblots (n=3) were quantified using Image Lab Software (v5.1) (Bio‑Rad Laboratories, Inc., assay revealed that cucurbitacin B may effectively reduce the Hercules, CA, USA). viability of U‑2 OS cells. The present study further analyzed whether cucurbitacin B reduced viability by inducing apop- Statistical analysis. The data are presented as mean ± stan- tosis. Annexin V/PI staining assay was performed to determine dard deviation, from three or six individual experiments. The apoptotic cell counts. Exposure to cucurbitacin B was identi- values were analyzed by one‑way analysis of variance followed fied to significantly increase the apoptotic cell counts (P<0.05; by Duncan's Multiple Range Test. All statistical analyses were Fig. 2), in a dose‑dependent manner with 100 µM concentra- performed using the SPSS software (version 22.0; IBM SPSS, tion exhibiting maximum effects. Armonk, NY, USA). P<0.05 was considered to indicate a Furthermore, Hoechst 33258 staining was performed to statistically significant difference. assess morphological changes in the U‑2 OS cells following cucurbitacin B treatment. Following treatment for 48 h, the Results chromatin of U‑2 OS cells was condensed and appeared to be brighter and deeper. Notably, treatment with cucurbitacin at Cucurbitacin B inhibits U‑2 OS cell proliferation. The viability higher concentrations of 80 and 100 µM demonstrated deeper of osteosarcoma cells following treatment with cucurbitacin B staining of nuclear chromatin than the lower doses (20 and was determined by MTT assay, and the findings are presented 40 µM). Highly condensed and stained chromatin indicates in Fig. 1. Osteosarcoma cells treated with different concen- characteristics of apoptotic cells, thus suggesting that cucur- trations of cucurbitacin B (20, 40, 80 and 100 µM) exhibited bitacin B effectively induced apoptosis in U‑2 OS cells. The reduced cell proliferation in a concentration‑dependent percentage of apoptotic cells increased significantly following manner. The viability percentage of U‑2 OS cells treated with cucurbitacin B treatment (20, 40, 80 or 100 µM) compared cucurbitacin B at 20 µM was 83%, whereas following the with the control (P<0.05; Fig. 3). 100 µM dose, the cell viability significantly decreased to 21% (P<0.05; Fig. 1). Cucurbitacin B inhibits migration of U‑2 OS cells. Following incubation with cucurbitacin B (25, 50 and 100 µM) for 24 h, Influence of cucurbitacin B on cell viability counts and the area of the wound was measured. In the cells that were not morphological changes in U‑2 OS cells. The results of MTT treated with cucurbitacin B, the distance between the scratches 808 ZHANG et al: CUCURBITACIN B INDUCES APOPTOSIS OF HUMAN OSTEOSARCOMA CELLS

Figure 3. Influence of Cucurbitacin B on the viability of U‑2 OS cells under Hoechst staining. Hoechst staining using a fluorescence microscope (Nikon Eclipse TiS coupled with NIS‑Elements imaging software) revealed a significantly increased number of cells under apoptosis following cucurbitacin B treatment. *P<0.05 vs. control; #P<0.05 vs. 20 µM. Data are presented as mean ± standard deviation, n=3. OS, osteosarcoma.

Figure 4. Influence of cucurbitacin B on cell migration determined by a wound healing assay. Cucurbitacin B exposure significantly reduced cell migration of the U‑2 OS cells over the wound area in a dose‑dependent manner. *P<0.05 vs. control; #P<0.05 vs. 25 µM. Data are presented as mean ± standard deviation, n=6. OS, osteosarcoma. Figure 5. Cucurbitacin B reduces activities of MMP‑2, MMP‑9 and VEGF in U‑2 OS cells. This was determined by (A) gelatin zymography following exposure to cucurbitacin B, indicating marked downregulation in the expressions of MMP‑2 and ‑9, (B) quantified results from the gelatin zymography was significantly reduced when compared with U‑2 OS cells indicating a significant downregulation of MMP activity. (C) Western blot that were exposed to cucurbitacin, in a dose‑dependent manner analysis indicates a marked reduction in the expression of MMP‑2, MMP‑9 * # (P<0.05). In the OS cells that were incubated with cucurbit- and VEGF. P<0.05 vs. control; P<0.05 vs. 25 µM. Data are presented as mean ± standard deviation, n=3. MMP, matrix metalloproteinases; VEGF, acin B the cell free area was evidently increased, the distance vascular endothelial growth factor; OS, osteosarcoma; L1, control; L2, increased with concentration of cucurbitacin B (Fig. 4). The 20 µM cucurbitacin B; L3, 40 µM cucurbitacin B; L4, 80 µM cucurbitacin B; growth and movement of the cells in the cell free scratch L5, 100 µM cucurbitacin B. area observed in cells that were not treated with cucurbitacin suggests cell division and migration were occuring, which were supressed following cucurbitacin B treatment. when compared with higher doses, 50 and 100 µM. Furthermore, marked suppression of VEGF levels was also observed following Cucurbitacin B downregulates MMPs. It has been established cucurbitacin B exposure in a dose‑dependent manner (Fig. 5C). that MMPs principally contribute to the invasion and metastasis These observations suggest that cucurbitacin B was able to of tumor cells (36‑38). In the present study, to further assess markedly supress cell migration and inhibit angiogenesis, whether cucurbitacin inhibits MMPs, the expression of MMP‑2 potentially by downregulating the expression of MMP2, MMP9 and ‑9 were determined by gel zymography and western and VEGF. blot analysis. The result of the current study indicated that cucurbitacin B exposure at all assessed concentrations caused Influence of cucurbitacin B on the expressions of apoptotic marked inhibitions in the expressions of MMPs (Fig. 5A). pathway proteins. Apoptosis is a coordinated network of The zymography analysis revealed significantly supressed genes that lead to cell death and is the target pathway in many activities of MMP‑2 and ‑9 in a dose‑dependent manner anticancer therapies. Anticancer drugs are known to induce (Fig. 5B). Furthermore, western blot analysis revealed a apoptosis by targeting cells that harbour genetic damage or that markedly decreased expression level of MMP2 and MMP9 in divide inappropriately (39). The present study assessed the influ- cucurbitacin B‑treated cells. However, relatively little inhibition ence of cucurbitacin B on the expression of apoptotic pathway was observed following treatment with 25 µM cucurbitacin proteins in U‑2 OS cells to investigate the molecular events EXPERIMENTAL AND THERAPEUTIC MEDICINE 14: 805-812, 2017 809

Figure 7. Cucurbitacin B downregulates the expression of MAPK signalling cascades. Cucurbitacin B exposure markedly inhibited the activation of JNK, ERK1/2 and p38, thus inhibiting the signaling pathway, demonstrated by western blot analysis. MAPK, mitogen activated protein kinases; JNK, c‑Jun N‑terminal kinases; ERK, extracellular signal‑regulated kinases; p‑JNK, phosphorylated‑JNK; p‑ERK, phosphorylated ERK; L1, control; L2, 25 µM cucurbitacin B; L3, 50 µM cucurbitacin B; L4, 100 µM cucurbitacin B.

antagonist killer (Bak) and Bad] (43). Cucurbitacin B exposure resulted in a multi‑fold increase in the Bad and Bax protein expression (P<0.05; Fig. 6B) with significant downregulation of Bcl‑2 and Bcl‑xL expression (P<0.05; Fig. 6B). The upregulated expression of Bax and Bad indicates the activation of the apoptotic pathway. Therefore, with enhanced levels of Figure 6. Cucurbitacin B modulates the expression of apoptosis pathway proteins. Cucurbitacin B caused effective upregulation of caspase‑3, ‑8 pro‑apoptotic proteins, cucurbitacin B aids in elevated levels and ‑9 in addition to an increase in the expression of Bax and Bad, while of caspases, which eventually induces apoptosis. suppressing the expression of Bcl‑2 and Bcl‑xL, demonstrated in (A) western * # blot analysis and (B) quantification of the results. P<0.05 vs. control; P<0.05 Cucurbitacin modulates the MAPK signalling cascades. It vs. 100 µM. Data are presented as mean ± standard deviation, n=3. Bcl‑2, B‑cell lymphoma 2; Bcl‑xL, Bcl‑2 extra large; Bad, Bcl‑2‑associated death has been well documented that activation of MAPK pathways promoter; Bax, Bcl‑2 associated X; L1, control; L2, 25 µM cucurbitacin B; serves a critical role in inhibiting apoptosis. In the present L3, 50 µM cucurbitacin B; L4, 100 µM cucurbitacin B. study, in order to elucidate the mechanisms underlying cucurbitacin B‑induced apoptosis, the expression levels of MAPK cascade proteins, JNK, p‑JNK, p38, p‑p38, ERK1/2, associated with cucurbitacin‑induced cell death. A significant and p‑ERK1/2 were analyzed by western blot analysis. The increase in the expression of caspase‑3, ‑8 and ‑9 in the U‑2 OS results demonstrated an enhanced expression of the proteins cells exposed to cucurbitacin B was observed following western in the U‑2 OS cells that were not exposed to cucurbitacin blot analysis (P<0.05; Fig. 6). The activation of caspase‑9 and ‑8 (Fig. 7). However, U‑2 OS cells treated with cucurbitacin B have previously been documented, suggesting the involvement exhibited marked downregulation in the phosphorylated levels of intrinsic and extrinsic apoptotic pathways that subsequently of ERK1/2, p38 and JNK. Whereas substantial decreases were lead to the activation of caspase‑3 (40). Therefore, in the present observed in the expression levels of p38 and ERK1/2, JNK study the enhanced expression of caspases by cucurbitacin B and p‑JNK levels were decreased to a lesser extent. However, indicates an increased level of apoptosis in U‑2 OS cells. marked inhibitions were observed following cucurbitacin B Furthermore, the expression of Bcl‑2 family members was treatment at all the three assessed concentrations, with 100 µM also assessed under the influence of cucurbitacin B. The Bcl‑2 exerting maximum effects. The results indicated that the family are key proteins in the regulation of intrinsic mito- downregulation of the MAPK signalling pathway may have chondrial pathways of apoptosis, specifically controlling the aided in induction of apoptosis of the U‑2 OS cells. release of cytochrome c, which further regulates the caspases cascade (41,42). The Bcl‑2 family comprises anti‑apoptotic Effects of cucurbitacin B on the JAK2/STAT3 signal‑ (Bcl‑2 and Bcl‑xL) and pro‑apoptotic proteins [Bax, Bcl‑2 ling pathway. The JAK/STAT3 pathway serves a pivotal 810 ZHANG et al: CUCURBITACIN B INDUCES APOPTOSIS OF HUMAN OSTEOSARCOMA CELLS

Cucurbitacin B, which is one of the most abundant cucurbitacins (27) exhibits anti‑inflammatory activity and is used traditionally to treat hepatitis (28,48). Previous studies have demonstrated the antitumor activities of cucurbitacin B in various human cancer cell lines and tumor xenografts (49,50). However, the mechanisms underlying the anticancer activities are yet to be elucidated. Studies are currently focussing on the identification and development of small molecules that inhibit major cell signalling pathways, as an important strategy in anticancer drug research (51‑53). The present study investi- gated the effects of cucurbitacin B on cell proliferation and the MAPK and JAK2/STAT3 pathways in U‑2 OS cells. Inhibition of cell proliferation is an important indicator of anticancer activity. Treatment with cucurbitacin B (20‑100 µM) exhibited evident growth inhibition. Furthermore, cucurbitacin B markedly induced the apoptosis of U‑2 OS, as observed by Hoechst staining and flow cytometry analysis followed by Annexin V/PI staining. The events underlying the induction of apoptosis were also analysed. Apoptosis occurs via the intrinsic mitochondrial pathway and the extrinsic membrane death receptor pathways. Cucurbitacin B exposure caused marked activation and enhanced expression of caspase‑9, ‑8 and ‑3. These observations suggest that cucurbitacin B induces apoptosis via the intrinsic pathway, which is in accordance with a previous study using chemotherapeutic drugs as gallic acid (54). Furthermore, Liu et al (55) reported that the majority of chemotherapeutic drugs were able to induce apoptosis through the intrinsic mitochondrial pathway. Figure 8. Cucurbitacin B downregulates the expression of JAK2/STAT3 signalling cascades. (A) Western blot analysis indicates that cucurbitacin B The mitochondria‑dependent apoptotic pathway is induced marked downregulation of JAK2 and STAT3 expression, thereby governed by Bcl‑2‑family proteins that includes pro‑apoptotic inhibiting the signaling cascade and (B) quantified results indicate a signifi- (BH3‑interacting domain death agonist, Bax, and Bak) and * cant reduction in expression of JAK2/STAT3 signaling cascades. P<0.05 anti‑apoptotic (Bcl‑2 and Bcl‑xL) proteins (56,57). Bax vs. control; #P<0.05 vs. 100 µM. Data are presented as mean ± standard deviation, n=3. JAK2, Janus kinase 2; STAT3, signal transducer and activator promotes apoptotic factors and induces apoptosis, whereas of transcription 3; L1, control; L2, 25 µM cucurbitacin B; L3, 50 µM cucur- Bcl‑2 inhibits the release of pro‑apoptotic proteins (58). bitacin B; L4, 100 µM cucurbitacin B. Cucurbitacin B notably enhanced the expression of Bad and Bax and supressed the levels of Bcl‑xL and Bcl‑2 indicating activation of the intrinsic apoptotic cascade. role in the transduction of a multitude of signals that are Cancer metastasis presents a huge challenge in cancer critically involved in the development and homeostasis in therapy and thus, blocking cancer cell metastasis is an mammals (44). STAT3 has been reported to be constitutively important approach. MMPs serve crucial roles in metastasis, activated in different types of cancer in humans (45). It has angiogenesis and also cause the release of growth factors from been demonstrated that the inhibition of STAT3 leads to the extra cellular matrix (59). MMP‑2 and ‑9 are well docu- apoptosis and inhibition of cancer cell proliferation (46). mented to be associated with the invasive metastatic potential In the present study, western blot analysis was performed of tumor cells (60). In U‑2 OS cells a markedly enhanced to assess the expression level of phosphorylated forms of expression of MMP‑2 and ‑9 was observed. Cucurbitacin B JAK2 and STAT3 following exposure to cucurbitacin B. supressed the levels of MMP‑2 and ‑9 at all the tested doses The results of the current study indicate that cucurbitacin B and inhibited the activities of the enzymes, thus exhibiting treatment significantly reduced the levels of activated JAK2 anti‑metastatic activity. The observations of the cell migration and STAT3 (P<0.05; Fig. 8). Cucurbitacin B at 100 µM assay also reveal the potent effects of cucurbitacin B on the concentration caused a multi‑fold decrease in the expression inhibition of cell migration and therefore, metastasis. of STAT3 and JAK2. Furthermore, the inhibition of STAT3 The JAK/STAT3 pathway is one of the major pathways and JAK2 expression was dose‑dependent. Therefore, these that regulate and control various vital physiological processes. observations indicate that cucurbitacin significantly inhibited JAK activation exerts critical roles in cell proliferation, the STAT3/JAK2 pathway and this may have a part in the differentiation, migration and apoptosis (44,61). Activated increase rate of apoptosis observed. JAKs phosphorylate cellular substrates, including the STAT family, which are vitally associated with oncogenic signalling Discussion pathways (62). Constitutive activation of STAT3 is typically observed in cancer cells and has been demonstrated to serve a Cucurbitacins are widely used in traditional Chinese medi- critical role in tumor cell growth and survival in human solid cines and are the bitter principles of Cucurbitaceae (25,47). tumors (62,63). STAT3 also upregulates anti‑apoptotic proteins EXPERIMENTAL AND THERAPEUTIC MEDICINE 14: 805-812, 2017 811 as Mcl‑1 and Bcl‑xL (63,64). Therefore, blocking the pathway 9. 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